Bottom Line:
Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity.These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity.The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

Affiliation: KOKORO-Biology Group and CREST-JST, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University Osaka, Japan.

ABSTRACTOlfactory sensory neuron (OSN) axons coalesce into specific glomeruli in the olfactory bulb (OB) according to their odorant receptor (OR) expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α) family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

Figure 2: Abnormal glomerular morphology in prenatal and neonatal PcdhaΔCR/ΔCR mice. (A) Ectopic glomerulus-like structures of M71-expressing OSNs were found in neonatal PcdhaΔCR/ΔCR mice by whole-mount observation. X-gal-stained lateral M71 glomeruli in whole-mounted OBs from WT (a, b) or PcdhaΔCR/ΔCR(ΔCR/ΔCR) (c, d) mice at P0. In PcdhaΔCR/ΔCR mice, abnormal axonal projections from the olfactory nerve were often detected (arrowheads). Melanocytes (arrows) were visible on some of the whole-mount preparations of the olfactory bulbs. Scale bars, 500 μm. (B) Sectional analysis of the coalescence of WT (a–f) and PcdhaΔCR2/ΔCR2 (g–l) P2 axons on embryonic day (E) 17.5. Serial sections of OBs were double-labeled with anti-β-galactosidase (for P2, green) and anti-NCAM (red) antibodies. There were more P2 glomerulus-like structures (arrowheads) in the PcdhaΔCR2/ΔCR2 (ΔCR2/ΔCR2) mice (See Figure 3A). Scale bar, 100 μm. (C) OBs in WT (a–c) and PcdhaΔCR/ΔCR mice (g–i) were double-labeled with anti-NCAM (red) and anti-MAP2 (green) antibodies. Due to the orientation shown in panels (d) and (j), in PcdhaΔCR/ΔCR mice, the OSN axons appeared to extend beyond the normal confines of the glomerular layer and often terminated as an intensely stained spatially restricted and condensed structure (j and l, arrows). In addition, the primary axons terminated in less clearly defined glomeruli in PcdhaΔCR/ΔCR (ΔCR/ΔCR) than in WT (+/+) mice. Primary glomerular structures could be detected in WT (d, f, asterisks) but not PcdhaΔCR/ΔCR mice (j, l) (See Figures 6B,C). Immunostaining with anti-MAP2 (green) antibody did not show significant differences between WT (e) and PcdhaΔCR/ΔCR (k) mice. Scale bar, 100 μm.

Mentions:
As described above, the Pcdh-α-deficient mice had multiple, small, extraneous glomeruli for all the OSN axons examined. These ectopic glomeruli persist until adulthood (Hasegawa et al., 2008). To determine whether the abnormalities in the Pcdh-α-deficient olfactory system resulted primarily from an inability of homotypic OSN axons to coalesce, we next analyzed the formation of glomeruli during early development using mice from crosses between M71-IRES-taulacZ or P2-IRES-taulacZ and WT or PcdhaΔCR/ΔCR mice (Hasegawa et al., 2008). The glomerular structures are initiated on embryonic day (E) 15–16, when both OSN fibers and mitral cell dendrites contribute to the formation of glomerulus-like structures (Blanchart et al., 2006). In the PcdhaΔCR/ΔCR mice, we observed some stray marked fibers that projected to inappropriate regions distant from the target site, and many more M71 and P2 glomerulus-like structures than in WT mice, even on postnatal day 0 (P0) (arrowheads in Figures 2A, 3A).

Figure 2: Abnormal glomerular morphology in prenatal and neonatal PcdhaΔCR/ΔCR mice. (A) Ectopic glomerulus-like structures of M71-expressing OSNs were found in neonatal PcdhaΔCR/ΔCR mice by whole-mount observation. X-gal-stained lateral M71 glomeruli in whole-mounted OBs from WT (a, b) or PcdhaΔCR/ΔCR(ΔCR/ΔCR) (c, d) mice at P0. In PcdhaΔCR/ΔCR mice, abnormal axonal projections from the olfactory nerve were often detected (arrowheads). Melanocytes (arrows) were visible on some of the whole-mount preparations of the olfactory bulbs. Scale bars, 500 μm. (B) Sectional analysis of the coalescence of WT (a–f) and PcdhaΔCR2/ΔCR2 (g–l) P2 axons on embryonic day (E) 17.5. Serial sections of OBs were double-labeled with anti-β-galactosidase (for P2, green) and anti-NCAM (red) antibodies. There were more P2 glomerulus-like structures (arrowheads) in the PcdhaΔCR2/ΔCR2 (ΔCR2/ΔCR2) mice (See Figure 3A). Scale bar, 100 μm. (C) OBs in WT (a–c) and PcdhaΔCR/ΔCR mice (g–i) were double-labeled with anti-NCAM (red) and anti-MAP2 (green) antibodies. Due to the orientation shown in panels (d) and (j), in PcdhaΔCR/ΔCR mice, the OSN axons appeared to extend beyond the normal confines of the glomerular layer and often terminated as an intensely stained spatially restricted and condensed structure (j and l, arrows). In addition, the primary axons terminated in less clearly defined glomeruli in PcdhaΔCR/ΔCR (ΔCR/ΔCR) than in WT (+/+) mice. Primary glomerular structures could be detected in WT (d, f, asterisks) but not PcdhaΔCR/ΔCR mice (j, l) (See Figures 6B,C). Immunostaining with anti-MAP2 (green) antibody did not show significant differences between WT (e) and PcdhaΔCR/ΔCR (k) mice. Scale bar, 100 μm.

Mentions:
As described above, the Pcdh-α-deficient mice had multiple, small, extraneous glomeruli for all the OSN axons examined. These ectopic glomeruli persist until adulthood (Hasegawa et al., 2008). To determine whether the abnormalities in the Pcdh-α-deficient olfactory system resulted primarily from an inability of homotypic OSN axons to coalesce, we next analyzed the formation of glomeruli during early development using mice from crosses between M71-IRES-taulacZ or P2-IRES-taulacZ and WT or PcdhaΔCR/ΔCR mice (Hasegawa et al., 2008). The glomerular structures are initiated on embryonic day (E) 15–16, when both OSN fibers and mitral cell dendrites contribute to the formation of glomerulus-like structures (Blanchart et al., 2006). In the PcdhaΔCR/ΔCR mice, we observed some stray marked fibers that projected to inappropriate regions distant from the target site, and many more M71 and P2 glomerulus-like structures than in WT mice, even on postnatal day 0 (P0) (arrowheads in Figures 2A, 3A).

Bottom Line:
Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity.These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity.The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

Affiliation:
KOKORO-Biology Group and CREST-JST, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University Osaka, Japan.

ABSTRACTOlfactory sensory neuron (OSN) axons coalesce into specific glomeruli in the olfactory bulb (OB) according to their odorant receptor (OR) expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α) family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.